The present invention relates to generally to the fields of organic chemistry and biochemistry. In particular, the present invention is directed to compounds which release nitric oxide (NO) in a controlled manner, as well as methods for the preparation and use thereof.
Nitric oxide has been implicated in a variety of important bioregulatory processes [for a review, see Moncada, S. et al., "Nitric Oxide: Physiology, Pathophysiology, and Pharmacology," Pharmacolog. Rev. 43:109-142 (1991). For example, nitric oxide has been identified as the endogenous stimulator of the soluble guanylate cyclase; in this role, NO can be considered the endogenous nitrovasodilator and NO-dependent vasodilator tone is probably one of the most fundamental adaptive mechanisms in the cardiovascular system. In addition, NO is an effector molecule released by murine macrophages and other cells after immunological activation.
NO is synthesized from the amino acid L-arginine by an enzyme, NO synthase; the precise mechanism for the biosynthesis has not, however, been eludicated. It is believed that there are at least two forms of the enzyme: a constitutive form which releases NO for short periods in response to receptor or physical stimulation; and a second form which is induced after activation of macrophages, endothelial cells and certain other cells by cytokines and which, once expressed, synthesizes NO for extended periods.
The constitutive form of NO synthase is implicated in the transduction mechanism for soluble guanylate cyclase, and thus in one of the mechanisms whereby cells regulate their own function or communicate with others. In addition, the release of NO in the cardiovascular system acts as a general adaptive mechanism whereby the vascular endothelium responds to changes in its environment and regulates blood flow and blood pressure through an action on the vascular smooth muscle. NO also regulates the interaction between the endothelium and the platelets (and probably blood-borne cells); it may also play a role in the control of vascular smooth muscle proliferation. The NO released by the constitutive enzyme may also play regulatory roles in other cells; for example, it is known to be linked to the stimulation by the excitatory antino acids of specific receptors in the central nervous system, and it may participate in the regulation of the secretion or action of various hormones.
NO released after immunological stimulation by the other form of the enzyme as part of the host defense mechanism has been shown to be cytotoxic or cytostatic for tumor cells and invasive organisms. Further, some forms of local or systemic tissue damage associated with immunological conditions could prove to be related as well to the release of NO. NO may also play a role in the normal regulation of the response of cells to mitogens, or contribute to the cytotoxic actions of other cells that play a role in specific immunity.
An elucidation of the biological roles of NO is complicated by the fact that most of the details about the function, distribution, and interaction of the two forms of enzyme involved in the synthesis of NO have yet to be studied. NO is itself extremely poisonous and unstable in the presence of oxygen. It is a highly reactive gas in its pure form, attacking most metals and plastics, and can only be obtained in relatively low pressure cylinders. Moreover, NO has limited solubility in aqueous media, making it difficult to introduce reliably into most biological systems without premature decomposition. In view of the central importance of NO as both a transducer and as an effector molecule, however, it is apparent that agents for the controlled release of NO would be invaluable in the continued research on the roles of NO in human physiology and pathology. Moreover, the therapeutic potential of agents which may be employed to release NO in a controlled manner is enormous.
Various vasodilators are known which release NO either spontaneously or upon activation by chemical or enzyrnatic means. For example, the release of NO from molsidomine (N-ethoxycarbonyl-3-morpholinosydnonimine) begins with an enzymatic hydrolysis catalyzed mainly by liver esterases, followed by a pH-dependent ring opening reaction catalyzed by hydroxyl ions; a subsequent oxidative process is then essential for further decomposition and NO release [Bohn, H. and K. Schoenafinger, "Oxygen and Oxidation Promote the Release of Nitric Oxide from Sydnonimines," J. Cardiovasc. Pharmacol. 14(Suppl.11):S6-S12 (1989)]. The vasodilators glyceryl trinitrate and sodium nitroprusside release nitric oxide upon redox activation. Other agents, such as iron-sulfur cluster nitrosyls, decompose spontaneously to release NO [Flitney, F.W. et al., "Selective retention of iron-sulphur cluster nitrosyls in endothelial cells of rat isolated tail artery: association with protracted vasodilator responses, J. Physiol. 459:89P (1993)].
Agents of the structure XN(O-)N=O, where X is a nucleophile residue, have been proposed as vehicles for the controlled delivery of NO into a biological system [Maragos, C. M. et al., "Complexes of NO with Nucleophiles as Agents for the Controlled Biological Release of Nitric Oxide. Vasorelaxant Effects," J. Medicinal Chem. 34:3242-3247 (1991)]. By varying the structure of the nucleophile residue, compounds having a range of decomposition rates and extents of nitric oxide release were prepared; the half-lives at 37.degree. C. and pH 7.4 ranged from 2.1 to 39 minutes. In addition, the vasoactivity of these compounds was correlated with the quantity of NO generated under the test conditions. A series of compounds (in which X=Et.sub.2 N) prepared by reaction of the ion with a variety of electrophiles exhibited even greater stability; the products of these alkylations were found to be remarkably resistant to hydrolysis [Saavedra, J. E. et al., "Secondary Amine/Nitric Oxide Complex Ions, R.sub.2 N[N(O)NO].sup.-. O-Functionalization Chemistry," J. Org. Chem. 57:6134-6138 ( 1992)]. A similar range of half-lives in solution were observed with zwitterionic polyamine/NO adducts prepared by reaction of NO with polyamines; the products were stable in solid form [Hrabie, J. A. et at., "New Nitric Oxide-Releasing Zwitterions Derived From Polyamines," J. Org. Chem. 58:1472-1476 (1993); U.S. Pat. No. 5,155,137 to Keefer et al., the entire disclosure of which is hereby incorporated by reference]. While some of these compounds may have potential utility as prodrugs, they nonetheless either decompose spontaneously or are dependent upon chemical and/or enzymatic activation for release of NO. Thus, these compounds are essentially simple variants of the heretofore-known vasodilator compositions.
For a variety of experimental uses, it would be particularly advantageous if the release of NO from an agent could be substantially prevented until the agent is activated by the researcher, for example by illumination. It has been reported that release of nitric oxide from an iron-sulfur cluster nitrosyl (Roussin's 1858 "black" salt, or RBS) is accelerated by exposure to laser light [Boulton, C. L. et al., "Nitric Oxide induces transient synaptic depression in rat hippocampal slices," J. Physiol, Abstract C37, p. 51P (Leeds Meeting 1993); Flitney, F. W. et al., "Photochemical release of nitric oxide from iron-sulphur cluster nitrosyls: laser potentiation of vasodilator actions on rat isolated tail artery," J. Physiol. 459:90P (1993)]. Ruthenium nitrosyl chloride salts have also been used as agents for delivery of NO upon exposure to near-UV irradiation [Williams, J. H. et al., "Photolytic release of nitric oxide from ruthenium nitrosyl chloride depresses an APS-sensitive response but does not induce long-term potentiation in area CA1 of the rat hippocampus in vitro," J. Physiol., Abstract C19, p. 33P (Leeds Meeting 1993); Carter, T. D. et al., "Photochemical release of nitric oxide from ruthenium nitrosyl trichloride: relaxation of rabbit isolated aortic rings mediated by photorelease of nitric oxide," J. Physiol., Abstract C20, p. 34P (Leeds Meeting 1993). Unfortunately, these agents comprise metals which themselves may interfere with the physiological phenomena under investigation or may be toxic. Therefore, there remains a need for additional agents useful for the delivery of NO which do not suffer from the drawbacks of the heretofore-known compositions. Such compositions would be very useful in a number of fields, such as biochemistry, cell biology and neurobiology.
It is an object of the present invention to provide compositions for delivery of nitric oxide which remain stable until it is desired to release NO by a particular triggering means, as well as methods for the preparation and use thereof.